Simultaneous estimation of dimenhydrinate, cinnarizine and their toxic impurities benzophenone and diphenylmethylpiperazine; in silico toxicity profiling of impurities

The British Pharmacopeia (BP) reported that the carcinogenic and hepatotoxic, benzophenone (BZP) is a dimenhydrinate (DMH) impurity. On the other hand, cinnarizine (CIN) is reported to have five impurities (A–E). The toxicity profile of CIN impurities was studied and the in silico data revealed that impurity A [1-(diphenylmethyl)piperazine] (DPP) was the most toxic CIN impurity, and hence it was selected during this work. TLC-densitometric method was developed for separation and simultaneous quantitation of DMH, CIN and their toxic impurities. In the proposed method hexane : ethanol : acetone : glacial acetic acid (7 : 3 : 0.7 : 0.5, by volume) with UV scanning at 225 nm were used. Method validation was carried out according to ICH guidelines and linearity was achieved in the range 0.2–4, 0.5–5, 0.1–2.0, and 0.05–2.2 μg per band for DMH, CIN, BZP and DPP, respectively. On the application of the method to pharmaceutical formulation, no interference from additives was observed. The greenness of the method was evaluated using the analytical eco-scale and the results revealed the low negative environmental impact of the developed method.

Recently, providing all the necessary information about impurities and harmful effects on human health gained immense interest during the development of new drug molecules. The presence of these undesirable substances, even in triing quantity may affect the safety and potency of pharmaceutical products. 17 Toxicity proling of impurities is laborious, time-consuming, expensive, and of ethical concerns, from this point, many computational approaches for virtual toxicity proling were developed, they collect databases containing ADME and toxicities data to train physiologically-based pharmacokinetics model and toxicity predictions. 18 Nowadays, numerous in silico-ADMET/Tox sowares are widely used which saves time and cost.
The environmental consciousness increased during the last few decades leading to emerging of the green analytical chemistry concept. The main principles of this concept are the reduction or elimination of the use and the generation of hazardous substances during the whole analytical procedures. Various green chemistry metrics are developed to evaluate the greenness criteria of such procedures. Eco-scale is a semiquantitative tool proposed by Van Aken et al. 19 which is used to evaluate different analytical parameters: the amount of reagents, their hazards, energy, and waste. 20 Potentiometric and argentometric titration were recommended for the determination of DMH in BP 5 and EP, 21 respectively. For the same purpose, USP 22 described an HPLC method using a mobile phase of ammonium acetate: methanol (80 : 20 v/v) owed at a rate of 1.5 mL min À1 with UV detection at 229 nm. Additionally, BP 5 and European Pharmacopeia (EP) 21 developed a potentiometric titration method for estimation of CIN. However, the DMH and CIN combination is not officially included in any pharmacopeia.
A literature survey revealed that DMH was analyzed by spectrophotometric, 23  Benzophenone, was reported to have the highest toxicity among the DMH impurities. However, no available literature for the toxicity assessment of CIN impurities (A-E). Thus, this work aims to use PreADMET application for the rst time to expect the toxicity prole of CIN impurities (A-E). The TLCdensitometry is considered a sensitive, accurate, and reliable analytical tool that is cost and time effective. 46,47 Additionally, no method was reported for the simultaneous determination of DMH, CIN, and their impurities (BZP and DPP). Hence, the current work was also aimed to develop a TLC-densitometric for simultaneous determination of DMH, CIN, and their toxic impurities. The method validation was carried out following ICH guidelines. 48

Experimental
All experiments complies with theýrelevant laws and institutional guidelines of Beni-Suef University and approved byýthe Safety and Occupational Health Committee at Faculty of Pharmacy, Beni-Suef University.

Materials and reagents
Cinnarizine pure sample was obtained as a gi from Amoun Pharmaceuticals (Cairo, Egypt) with a purity of 100.26% according to the analysis results of the reported method, 45 DMH was kindly supplied from Kahira Pharmaceuticals and Chemical Industries Company (Cairo, Egypt) with a purity of 99.10% according to the reported method. 45 Benzophenone and DPP were purchased from Sigma Aldrich, Chemie GmbH (Darmstadt, Germany) with claimed purity of 97% and 99%, respectively according to the manufacturing certicates of analysis. Methanol, ethanol, acetone, hexane, methylene chloride, chloroform, and ethylacetate were of HPLC grade (Fisher, Loughborough, UK) and were purchased from Sigma Aldrich Chemie GmbH (Darmstadt, Germany). Orthophosphoric acid, glacial acetic acid, formic acid, triethylamine, and ammonia solution (33%) were bought from EL-Nasr pharmaceutical, Chemical Co., Abu Zabaal, Cairo, Egypt and were of analytical grade. Amocerebral® tablets, (batch no. 6221025030658) were manufactured by Amoun pharmaceutical company (Cairo, Egypt) each tablet was labeled to contain 20 mg of DMH and 10 mg of CIN.

Sowares
Toxicity proling was carried out using version 2.0 of Pre-ADMET online program soware. The toxicity proling includes testing of acute algae, daphnia and sh toxicity, Ames test for mutagenicity testing of several Salmonella typhimurium strains. Carcinogenicity testing is also included through 2 years carcinogenicity bioassay in rats and mice in addition to in vitro human ether-a-go-go related gene channel (hERG) inhibition testing. 49

Solutions
(a) Stock standard solutions of DMH, BZP, CIN, and DPP were prepared by accurate weighing of 0.1 g of each and dissolving in methanol to obtain a 100 mL stock solution of 1 mg mL À1 each.
(b) The stock solution of Amocerebral® tablets was prepared by weighing the content of ten tablets, then grinding them and mixing the resulted powder well. An amount of the tablets powder equivalent to 50 mg DMH and 25 mg CIN was weighed and transferred to a 25 mL volumetric ask. Methanol (10 mL) was then added and the solution was ultrasonicated for 30 min, cooled, the volume was adjusted with methanol and nally, the solution was ltered.

Instruments
Linomat V applicator with a 100 mL syringe was used to apply the samples. The TLC scanner model 3S/N (Camag, Muttenz, Switzerland) was used for scanning and the scanner was controlled with WinCATS soware (version 3.15). TLC aluminum plates (20 Â 10 cm) coated with 0.25 mm Silica gel 60 F254 (Merck, Darmstadt, Germany) were used as a stationary phase. Also, a UV lamp with a short wavelength of 254 nm (Vilber Lourmat, Cedex, France) was used during the optimization of the developed method.

Chromatographic conditions
TLC-densitometric method was conducted by applying the studied compounds to TLC plates (20 Â 10 cm with 250 mm thickness and 5 mm particle size) as bands of 3 mm width using Camag Linomat-V applicator. The bands were applied at 5 mm intervals and 15 mm from the bottom edge of the plate. Linear ascending development was performed to 8 cm in a chromatographic jar that was previously saturated for 30 min with a solvent mixture of hexane : ethanol : acetone : glacial acetic acid (7 : 3 : 0.7 : 0.5, by volume) at a temperature of 25 C and the separated bands were UV scanned at 225 nm.

Construction of calibration curves CIN, DPP, DMH and BZP
Different samples of DMH, BZP, CIN, and DPP in the range of 40-220, 10-200, 50-500, and 5-220 mg mL À1 were prepared in methanol in separate series of 10 mL volumetric asks using their respective stock solutions (1 mg mL À1 ). 10 mL of each sample was applied in triplicate to TLC plates. Ascending development was carried out following the instructions listed above.
The calibration curves were constructed by plotting area under the peak against the corresponding analyte concentrations from which the regression equations were created.

Application to the pharmaceutical formulation
Different dilutions of Amocerebral® tablets within the linearity ranges of the developed method were prepared in methanol using the previously prepared stock solution (2 mg mL À1 DMH and 1 mg mL À1 CIN). The instructions followed for the construction of calibration curves were applied and the previously computed regression equations were used to calculate the concentrations of the studied drugs in the prepared samples. Additionally, the accuracy of the developed method was tested using the standard addition technique.

Results and discussion
Nowadays, developing a method for determination of drugrelated impurities is attractive to many researchers especially when they exhibit certain toxicity. Although toxicity proling is the cornerstone of the drug development process, it requires a huge amount of time, energy, and cost. That is why the use of computational tools for toxicity prediction is rapidly grown.
To date, no available analytical method for the simultaneous determination of DMH, CIN, and their pharmacopeial impurities. Besides, no toxicity proling study was published for CIN impurities. Therefore, in the current work, the toxicity proling of CIN impurities was studied using PreADMET application in addition to developing and optimizing a TLC-densitometric method for the simultaneous determination of the quaternary mixtures (DMH, BZP, CIN, and DPP). The greenness of the developed method was evaluated using the analytical eco-scale.
The developed method has the advantages of being novel, green, time and cost-effective. Besides, it more sensitive and selective in comparison to the reported method. 45 Table 1.

In silico toxicity proling of cinnarizine impurities
Examination of the preADMET toxicity screening results for CIN and its impurities (A-E) are shown in Table 2. The results revealed that all impurities showed positive AMES mutagenicity to only one of salmonella strains except DPP which is a mutagen for both TA1535_10RLI and TA1535_NA strains. Moreover, DPP showed the highest toxicity against algae, daphnia, and sh. Additionally, all the impurities showed positive carcinogenicity for either rat, mice, or both. Also, all of them have a medium risk for hERG_ inhibition. From these results, we can conclude that DPP was the most toxic CIN impurity and hence it was selected as an example of CIN toxic impurities to be quantied using the developed TLC-densitometric method.

Optimization of the mobile phase composition
Several trials were performed to achieve complete separation among the four studied compounds. Trials started with ethanol: ethylacetate (8 : 2, 2 : 8, and 5 : 5, v/v) where the peaks of CIN, DMH, and BZP appeared with the solvent front in all these trials. Aer that, nonpolar solvents were tested in a trial to enhance the retention of these rapidly eluted compounds. Different ratios of hexane : ethanol, chloroform : ethanol, and methylene chloride : ethanol were checked and the best non polar solvent was hexane in the ratio of (7 : 3, v/v) where the retention of DMH, CIN and BZP was improved but unresolved peaks for DMH and CIN were observed, besides, DPP appeared near the baseline. On the other hand, lower volumes of hexane signicantly affected the separation of other components. The effect of mobile phase pH on the separation efficacy was then tested by separate addition of different volumes of formic acid, ammonia solution (33%), triethylamine, orthophosphoric acid, and glacial acetic acid to the optimum developing system. It was noticed that the addition of 0.5 mL glacial acetic acid was sufficient to improve the separation between CIN and DMH. Additionally, different volumes of acetone were tested (0.3-0.8 mL) to improve the R f value of DPP without affecting the separation among the four compounds. Finally, complete separation was achieved upon using a mobile phase mixture of hexane : ethanol : acetone : glacial acetic acid (7 : 3 : 0.7 : 0.5, by volume). 3.00 Â 10 À4 TA100_10RLI À À + + À À TA100_NA À À À À À À TA1535_10RLI + + À À + + TA1535_NA À + À À À À

Selection of detection wavelength.
To improve the sensitivity of the developed method, different scanning wavelengths were tried (215, 225, and 254 nm). The highest sensitivity with low noise for all the scanned components was observed on scanning at 225 nm as shown in Fig. 2. 3.2.2. Slit dimensions of scanning light beam. Different slit dimensions were checked and 3 Â 0.3 mm 2 was proved to be the slit dimension that provided the highest sensitivity for all the studied drugs.
Finally, suitable resolution and acceptable peaks were attained where the R f values were 0.18, 0.55, 0.68, and 0.82 for DPP, CIN, DMH and BZP, respectively. Fig. 2.

Methods validation
Method validation was carried out according to guidelines for method validation from the International Conference on Harmonization (2005). 48 It was performed to estimate selectivity, linearity, sensitivity, accuracy, and precision.  Table 3. 3.3.2. Accuracy and precision. Accuracy of the developed method was evaluated by analyzing different concentrations of DMH, BZP, CIN, and DPP in their linearity ranges following the instructions of the method and calculated as percentage recovery. Good results were obtained and are given in Table 3. Additionally, the standard addition technique was applied to amocerebral® tablets to assess the accuracy of the proposed Where A is the peak area of the analyte and k is the intercept. c The x coefficient (n) of the polynomial regression equation for BZP: A ¼ mx 2 + nx + k. Where A is the peak area of the analyte and k is the intercept. d The inter-day precision (n ¼ 9), average SD of three different concentrations repeated three times within one. e The inter-day precision (n ¼ 9), average SD of three different concentrations repeated three times on three successive days. This journal is © The Royal Society of Chemistry 2020 RSC Adv., 2020, 10, 37439-37448 | 37443 method, the good recoveries obtained suggested no interference from excipients ( Table 4). The precision was assessed by measuring the intra and interday variations. Intraday variation (repeatability) was calculated by analyzing separately three concentrations of CIN and DMH (0.6, 2.6, and 3.4 mg per band), DPP (0.1, 1.3, and 2 mg per band) and BZP (0.20, 1, and 1.5 mg per band). Samples were analyzed three times on the same day following the instructions of the method and results were expressed as RSD (%). Interday variations (intermediate precision) were conrmed by analyzing the previous concentrations on three consecutive days. Acceptable results and low RSD were obtained (Table 3).

Limits of detection and quantitation.
Sensitivity of the developed method was proved by calculation of the limits of detection (LOD) and quantitation (LOQ). They were calculated using the following equations: LOD ¼ 3.3 Â SD/slope and LOQ ¼ 10 Â SD/slope. The standard deviation (SD) was calculated from the lower part of the calibration curve, while the slope referred to the slope of the calibration curve for each analyte. The proposed method showed high sensitivity as demonstrated by LOD and LOQ values given in (Table 3) 3.3.4. Selectivity. Selectivity of the proposed method was proven by the complete separation between the four analytes shown in Fig. 2. Additionally, values of resolution and selectivity factors were within the acceptable limits ensuring complete separation among the four components (Table 5).
3.3.5. Robustness. Robustness is characterized as the method's ability to tolerate minor changes intended in system parameters such as changing the percentage of acetic acid (AE0.05 mL) and saturation time (AE5 min). The effect of these changes on R f value was calculated as the SD. It was found that changing in the studied parameters had no signicant effect on the R f values of the separated peaks (Table 6) 3.3.6. System suitability testing parameters. System suitability was evaluated by calculating the capacity factor (K 0 ), tailing factor (T), selectivity (a) and resolution (R s ) factors. All measured parameters (Table 5) were within acceptable ranges, 50 indicating the high selectivity of the method established and ensuring its validity.
3.3.7. Application of the method. The developed method was successfully applied to the available dosage form; amocerebral® tablet. The results presented in Table 4 revealed the suitability of the developed method for the simultaneous determination of DMH and CIN in their available dosage form without interfering from tablets excipients. Furthermore, the application of standard addition technique conrmed the accuracy of the method.

Greenness assessment
Green analytical chemistry is designed to eliminate or reduce the amount of toxic solvents produced and consumed daily worldwide. Eco-scale provides a semiquantitative tool for assessment of the greenness of an analytical procedure, which is preferable than other qualitative ones (NEMI, E factor ) considering the environmental consequences and the energy consumption during the life cycle of the analytical method. Eco-scale was used to assess and easily compare the proposed and reported methods greenness, the calculated ecoscale scores of the developed method was >75 meaning excellent green analysis. 51 (Table 7).

Statistical analysis
The results obtained by the proposed TLC-densitometric method for amocerebral® tablets were statistically compared to the reported method. 45 Using Student's t-test and variance ratio F-test at a 95% condence level and the values of the calculated t and F were less than the tabulated ones (Table 4), revealing no signicant difference with respect to accuracy and precision between the proposed method and the reported one.

Conclusion
Benzophenone was reported as a carcinogenic and hepatotoxic impurity of DMH. On the other hand, the in silico toxicity proling was studied for CIN impurities and results showed that DPP (impurity A of CIN) had the highest toxicity among all CIN impurities (A-E). Likewise, a novel, accurate and precise TLC-densitometric method has been developed for the simultaneous determination of DMH, CIN and their toxic impurities; BZP and DPP, respectively. The method was cost and time-effective as many samples can be performed simultaneously using small quantities of solvents. Additionally, the proposed method was validated according to ICH guidelines and all results were within acceptable limits. The validated method has been successfully applied for the estimation of the active drugs in their pure forms and in their commercial dosage form. Furthermore, analytical eco-scale was used for evaluation of the greenness of the method where the results proved that the method had a low negative environmental impact.

Funding
This research did not receive any specic grant from funding agencies in the public, commercial, or not-for-prot sectors.